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. 2022 May 28;23(1):404.
doi: 10.1186/s12864-022-08631-4.

miR-155 exerts posttranscriptional control of autoimmune regulator (Aire) and tissue-restricted antigen genes in medullary thymic epithelial cells

Pedro Paranhos Tanaka #  1 , Ernna Hérida Oliveira #  1 , Mayara Cristina Vieira-Machado  1 , Max Jordan Duarte  1 , Amanda Freire Assis  1 , Karina Fittipaldi Bombonato-Prado  2   3 , Geraldo Aleixo Passos  4   5   6
Affiliations

miR-155 exerts posttranscriptional control of autoimmune regulator (Aire) and tissue-restricted antigen genes in medullary thymic epithelial cells

Pedro Paranhos Tanaka et al. BMC Genomics. .

Abstract

Background: The autoimmune regulator (Aire) gene is critical for the appropriate establishment of central immune tolerance. As one of the main controllers of promiscuous gene expression in the thymus, Aire promotes the expression of thousands of downstream tissue-restricted antigen (TRA) genes, cell adhesion genes and transcription factor genes in medullary thymic epithelial cells (mTECs). Despite the increasing knowledge about the role of Aire as an upstream transcriptional controller, little is known about the mechanisms by which this gene could be regulated.

Results: Here, we assessed the posttranscriptional control of Aire by miRNAs. The in silico miRNA-mRNA interaction analysis predicted thermodynamically stable hybridization between the 3'UTR of Aire mRNA and miR-155, which was confirmed to occur within the cellular milieu through a luciferase reporter assay. This finding enabled us to hypothesize that miR-155 might play a role as an intracellular posttranscriptional regulator of Aire mRNA. To test this hypothesis, we transfected a murine mTEC cell line with a miR-155 mimic in vitro, which reduced the mRNA and protein levels of Aire. Moreover, large-scale transcriptome analysis showed the modulation of 311 downstream mRNAs, which included 58 TRA mRNAs. Moreover, miR-155 mimic-transfected cells exhibited a decrease in their chemotaxis property compared with control thymocytes.

Conclusion: Overall, the results indicate that miR-155 may posttranscriptionally control Aire mRNA, reducing the respective Aire protein levels; consequently, the levels of mRNAs encode tissue-restricted antigens were affected. In addition, miR-155 regulated a crucial process by which mTECs allow thymocytes' migration through chemotaxis.

Keywords: Aire; Medullary thymic epithelial cell; Posttranscriptional control; Transwell migration; miR-155.

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Conflict of interest statement

Authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Prediction of posttranscriptional interaction between Aire mRNA 3’UTR and miR-155 as evaluated by RNA-Hybrid program. The interaction resulted in a significant minimal free energy (− 22 mfe) forming thus a stable hybrid structure
Fig. 2
Fig. 2
Posttranscriptional interaction between miR-155 with Aire 3’UTR was assayed by luciferase reporter gene assay (LRGA). pMIR-Aire-wt-3’utr (wild type 3’UTR) or pMIR-Aire-mut-3’utr (mutant 3’UTR) luciferase vector constructs were transfected into human HEK-293 T cells to demonstrate the possibility of occurrence of the miRNA-mRNA interactions within the cell milieu. The mutant 3’UTR was used to demonstrate the need of sequence specificity for the interaction to occur. Data presented are from three independent experiments, and standard error mean (SEM)
Fig. 3
Fig. 3
Relative expression of miR-155 in mTEC 3.10 cell line as detected by RT-qPCR. The mTEC cells were transfected (or not) with miR-155-5p mimic, causing significant increase in the levels of this miRNA 12 h after transfection. Data presented are from three independent experiments, and standard error mean (SEM). Difference between groups was analyzed by Student-t test, comparing data from control (not transfected) versus transfected cells (**P < 0.01)
Fig. 4
Fig. 4
Relative expression of Aire mRNA in mTEC 3.10 cell line as detected by RT-qPCR. The mTEC cells were transfected (or not) with miR-155-5p mimic, causing significant decrease in the levels of this mRNA 24 h after transfection. Data presented are from three independent experiments, and standard error mean (SEM). Difference between groups was analyzed by Student-t test, comparing data from control (not transfected) versus transfected cells (***P < 0.001)
Fig. 5
Fig. 5
Expression of AIRE protein in mTEC 3.10 cell line as detected by western-blot. The mTEC cells were transfected (or not) with miR-155-5p mimic, causing significant decrease in the levels of this protein. A western-blot detection of AIRE after 24 h of transfection. The GAPDH protein was used as housekeeping and to normalize data. The lanes/bands showed were cropped from the blot displayed in the Supp. Figure 1. B Bar graph resulted from quantification of western-blot bands show the effect of miR-155-5p mimic transfection on AIRE expression. The data are presented as the means and standard error of mean (SEM) from three independent determinations. Difference between groups was analyzed by Student t-test, comparing control (not transfected) versus transfected cells (**P < 0.01)
Fig. 6
Fig. 6
Expression of AIRE protein in mTEC 3.10 cell line as detected immunofluorescence. Immunofluorescence detection of AIRE (red dots in the nuclei stained in blue) after 24 h of transfection. The mTEC cells were not-transfected (A) or transfected (B) with miR-155-5p mimic. Transfection caused decrease in the protein intensity signal but not in the number of cells expressing AIRE
Fig. 7
Fig. 7
Transcriptome (mRNAs) expression profiles of mTEC 3.10 cell line transfected (or not) with miR-155-5p mimic. The mTEC cells total RNA samples were analyzed through microarray hybridizations, which allowed identify the differentially expressed mRNAs including those that encode tissue-restricted antigens (TRAs) or cell migration molecules. The dendrograms and heat-maps were obtained using the cluster and tree view algorithm considering 1.5-fold-change and 0.05 false discovery rate. Data were collected from three independent hybridizations (biological replicates). Heat-map legend: red = upregulation, green = downregulation, black unmodulation (Pearson’s correlation metrics)
Fig. 8
Fig. 8
Functional annotation for modulated (up or downregulated) mRNAs in mTEC 3.10 cells. The functional categories were identified by using DAVID genome database platform according to GO processes: biological processes (BP), cellular components (CC) and molecular function (MF). The rank is based on the enrichment score, which represents mean p-value. Only those mRNA-groups yielding < 0.05 Benjamini corrected p-value and containing at least three mRNAs are considered to be significant
Fig. 9
Fig. 9
Decrease of thymocyte migration towards mTEC 3.10 cells (chemotaxis) following miR-155-5p mimic transfection. Differences in the numbers of migrated thymocytes through porous membrane in conditioned medium from transfected or not transfected cells were quantified, and data are shown in bar graph form. The migration degree is plotted as migration index, in which values correspond to mean ± s.d. Data are representative of three independent experiments and were significantly different between control cells versus cells transfected with miR-155-5p mimic. (**P < 0.01, Student t-test)
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References

    1. Sansom SN, Shikama-Dorn N, Zhanybekova S, Nusspaumer G, Macaulay IC, Deadman ME, et al. Population and single-cell genomics reveal the Aire dependency, relief from Polycomb silencing, and distribution of self-antigen expression in thymic epithelia. Genome Res. 2014;24(12):1918–1931. doi: 10.1101/gr.171645.113. - DOI - PMC - PubMed
    1. Passos GA, Speck-Hernandez CA, Assis AF, Mendes-da-cruz DA. Update on Aire and thymic negative selection. 2017;153(1):10–20. 10.1111/imm.12831. - PMC - PubMed
    1. Passos GA, Genari AB, Assis AF, Monteleone-Cassiano AC, Donadi EA, Oliveira EH, et al. The thymus as a mirror of the body’s gene expression. In: Passos GA, et al., editors. Thymus transcriptome and cell biology. Cham: Springer-Nature; 2019. pp. 215–234.
    1. Klein L, Kyewski B, Allen PM, Hogquist KA. Positive and negative selection of the T cell repertoire: what thymocytes see (and don’t see) Nat Rev Immunol. 2014;14(6):377–391. doi: 10.1038/nri3667. - DOI - PMC - PubMed
    1. St-Pierre C, Trofimov A, Brochu S, Lemieux S, Perreault C. Differential features of AIRE-induced and AIRE-independent promiscuous gene expression in Thymic epithelial cells. J Immunol. 2015;195(2):498–506. doi: 10.4049/jimmunol.1500558. - DOI - PubMed